Inverse association of colorectal cancer prevalence to serum levels of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in a large Appalachian population

While incidence and mortality rates of colorectal cancer (CRC) have declined during the past decade, CRC remains the third most common cancer in both men and women and the third leading cause of cancer-related mortality in the United States [1, 2]. Major risk factors for CRC include age, a family or personal history of CRC, colorectal polyps, chronic inflammatory bowel disease, and inherited genetic alterations, such as familial adenomatous polyposis or hereditary nonpolyposis CRC (Lynch syndrome) [2, 3]. CRC rates are higher in men and in African American populations [2, 3]. Certain lifestyle-related factors also increase risk for CRC, including physical inactivity, obesity, smoking, and a diet high in red and processed meats [2, 3]. Recent cohort studies suggest constipation may also increase CRC risk [4, 5]. Conversely, a growing body of evidence suggests that use of aspirin and other anti-inflammatory medications [6‐9] and certain dietary supplements (e.g., calcium) may be protective against CRC [2]. In addition, the role of peroxisome proliferator-activated receptor-γ (PPAR-γ) in adipocyte differentiation, the antiproliferative and/or differentiating effects of PPAR-α and PPAR-γ ligands in human colon and other tumor cell lines, and the anticancer effects of both PPAR isotypes in animal models of CRC support a chemoprotective role for these nuclear hormone receptors [10, 11].

Certain environmental contaminants have also been linked to increased risk for incident CRC, including drinking water nitrate [12] and chloroform levels [13]. However, the link between CRC and other widespread contaminants, including perfluorocarbon compounds (PFC’s), remains unclear [14, 15]. Given the documented protective role of non-steroidal anti-inflammatory medications [6], and the growing number of studies supporting a role for PPARs in CRC prevention and treatment [10, 11], it is possible that certain perfluoroalkyl acids (PFAAs), including the widespread pollutants, perfluoroctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), may also be associated with reduced CRC risk. These compounds are potent PPAR ligands, and have demonstrated anti-inflammatory effects in vitro [16] and in animal studies [17] that are thought to operate via both PPAR-dependent and –independent pathways [18]. To date, only two studies in the same cohort of fluorochemical and film plant employees have assessed the association of PFAAs or any other PFCs to CRC: a survey study of 1400 workers [14] and an overlapping analysis of health claims data from 1301 employees [15]. While neither study documented significant associations between PFOS and CRC, conclusions were limited by very small numbers (N = 12 confirmed CRC cases), reliance on self-report or claims data, and lack of information on PFOS blood levels or potential confounders.

In this study, we investigated the association of prevalent colorectal cancer to PFOA and PFOS in a large Appalachian population who were exposed to elevated levels of PFOA through contaminated drinking water.

The study flow diagram is given in Figure 1. Participants who had received a diagnosis of cancer other than primary colon or rectal cancer (N = 4116) were excluded from the analytic sample, leaving a total of 49,312 eligible adults. Exclusion of participants with missing data on PFOA and PFOS (N = 296, 0.6%) and other covariates of interest (N = 1584, 3.2%) and of colorectal cancer cases that could not be validated due to missing information (N = 73) yielded a final study sample of 47,359, including 47,151 cancer-free controls and 208 adults with a medical record-confirmed diagnosis of primary colon or rectal cancer. Among validated CRC cases with information on diagnosis date (N = 193), 99 were diagnosed prior to 2000, and 94 received a diagnosis in 2000 or later (median 1999, range 1966 to 2006). Compared to participants included in the analyses, those with missing data on any covariate were more likely to be female and postmenopausal, to be older, less educated and heavier, and to report lower income and higher prevalence of comorbid medical conditions (P < 0.01).

The distribution of study population characteristics by CRC diagnosis is given in Table 1. Participants ranged in age from 21 to 105 years of age (mean (SD) = 45.71 (15.0) years), and were predominantly (97%) non-Hispanic white. Fifty-two percent were female, 38% reported an annual household income of less than $30,000, and 53% had received only 12 years of schooling or less. Sixty-three percent were employed, and 7% were disabled. Over 70% of the adults in this Appalachian population were overweight (BMI 25 or greater), and over 35% were obese (BMI ≥30).

Of the 47,359 eligible participants with no missing data, 208 were diagnosed with CRC. After adjustment for all other factors in the table, CRC retained significant, positive associations with age (p < 0.00001) and male gender (p < 0.05), and with postmenopausal status in women (P <0.003). Participants who were disabled or had been diagnosed with at least one chronic medical condition were also significantly more likely to have been diagnosed with CRC (P < 0.02). Conversely, those who were on lipid-lowering medications were less likely to have received a diagnosis of CRC (P <0.02).

Serum values of PFOA were elevated in this population, averaging 86.6 (255.1) ng/mL (median = 27.9, range <0.5-22,412 ng/mL), compared to a geometric mean of 3.7-4.2 ng/mL across adult age ranges in the 2003–2004 NHANES population [24]. In contrast, PFOS serum levels in our study sample were similar to those in the general U.S. population [24], ranging from <0.5 to 759.2 ng/mL and averaging 23.4 ± 16.3 ng/mL (median = 20.2 ng/mL). Likewise, serum levels of other PFAA’s for which adequate data were available were comparable to general background levels in the U.S [24, 25].

Table 2 details the associations between CRC diagnosis and serum levels of PFOA and PFOS. PFOS showed a strong, inverse association to diagnosis of CRC in both the minimally adjusted analysis and the full models. Those testing in the highest PFOS quartile were 80% less likely to have been diagnosed with CRC than those in the lowest quartile (odds ratio (OR) = 0.2, 95% confidence interval (CI) 0.1, 0.3, P for trend < 0.00001) after adjustment for age and BMI. Further adjustment for sociodemographic characteristics, menopausal status and hormone replacement therapy (HRT) use, lifestyle factors, and comorbidity did not materially alter these risk estimates (OR’s = 0.35 (CI 0.2, 0.5), 0.3 (CI 0.2, 0.5), and 0.2 (CI 0.2, 0.3) for the second, third, and highest quartile of PFOS, respectively; P for trend < 0.00001), nor did additional adjustment for gastrointestinal (GI) symptoms and for serum levels of folate, estradiol, cholesterol, uric acid, and C-reactive protein. The association of CRC to serum PFOS analyzed as a continuous variable showed a similar pattern, with the strength of the linear relationship remaining constant after adjustment for other demographic, lifestyle, and health-related factors (P < 0.00001, Table 2).

PFOA also demonstrated a significant, inverse, although more modest relationship to CRC (Table 2). Participants in the highest PFOA quartile showed an approximately 40% reduced likelihood of CRC diagnosis (age-adjusted OR = 0.6, CI 0.4, 0.9, P for trend = 0.002), although the linear trend was weak and non-significant when PFOA was analyzed as a continuous variable (p = 0.42). Additional adjustment for other demographic, socioeconomic, lifestyle, and health-related factors did not appreciably alter the strength or magnitude of this inverse association.

Further adjustment for other PFAAs, for anemia, or for diagnosed osteoarthritis, rheumatoid arthritis, and fibromyalgia or did not alter these associations, nor did exclusion of those with low hemoglobin levels (N = 2391, including 41 CRC cases).

As detailed in Tables 3, 4 and 5, the protective association of PFOA was more pronounced in men than in women (age-adjusted OR’s, respectively for the highest vs. the lowest quartile = 0.5 (CI 0.3, 0.9) vs. 0.8 (CI 0.4, 1.3), p for interaction < 0.05) and tended to be stronger in leaner (BMI < 30) than in obese adults (BMI 30+) (fully adjusted OR’s, respectively, for highest vs. lowest quartile = 0.5 (CI 0.3, 0.9) vs. 0.9 (CI 0.5, 1.8), p for interaction < 0.09). Similarly, the inverse relation of PFOA to prevalent CRC was significantly stronger in those diagnosed within the previous 6 years (2000 or later) relative to those diagnosed earlier (adjusted OR’s, respectively for the highest vs. the lowest quartile = 0.4 (CI 0.3, 0.7) vs. 0.9 (CI 0.5, 1.6)). The association of PFOS to likelihood of CRC was also more pronounced in those diagnosed more recently (adjusted OR’s, respectively, for highest vs. lowest quartile = 0.1 (CI 0.1, 0.2) vs. 0.4 (CI 0.3, 0.7)) (Table 5). We did not find evidence for a modifying effect of gender or BMI on the relation of CRC to PFOS, or of age or CRC treatment method on the association of CRC to either PFOA or PFOS.

Similarly, as illustrated in Table 6, restricting the analysis to those who had lived at the same residence since 1990–1995 or before and to CRC cases diagnosed in 2000 or later strengthened the inverse associations with both PFAAs. Adjusted OR’s for highest vs. lowest quartile were 0.1 (CI 0.1, 0.2) for PFOS and 0.4 (CI 0.2, 0.5) for PFOA. Further restricting CRC cases to those diagnosed in 2005–6 (N = 15 CRC cases) yielded similar findings (adjusted OR’s for highest vs. lowest quartile = 0.1 (CI 0.0, 0.5) for PFOS and 0.4 (CI 0.1, 1.4) for PFOA).

To our knowledge, this is the first large, community-based study to examine the potential links between CRC and environmental PFAAs, and the first to examine in detail the association of CRC to PFOA and PFOS. Our findings indicate a significant inverse association between both PFOS and PFOA and diagnosis of CRC in this large cross-sectional study. This association between PFOS and CRC was particularly pronounced, with a strong linear inverse relationship that remained robust even at lower exposure levels. After adjustment for multiple potential confounders, those with serum PFOS levels in the highest quartile were approximately 80% less likely to have received a diagnosis of CRC; likewise, those in the highest quartile of PFOA were approximately 40% less likely to have been diagnosed with CRC. These inverse associations were stronger with CRC diagnosed within the previous 6 years and in long term residents of the affected water districts. The relationship between PFOA and CRC was also more pronounced in men and leaner adults, and showed a strong linear trend only at lower serum concentrations comparable to those observed in the general US general population, suggesting a possible threshold or saturation effect for this PFAA.

Experimental studies have indicated carcinogenic effects of both PFOA and PFOS in rodent models, at least at high exposures; however, no associations between these PFAA’s and CRC have been reported. Moreover, published studies regarding potential links between PFAAs and cancer in humans are sparse and have yielded inconclusive findings. Two European studies, including a large Danish nested cohort study (N = 1240 liver, bladder, pancreatic, and prostate cancer patients) [26] and a small Greek case–control study (N = 40 cancer patients) [27], reported no associations between serum levels of either PFAA and cancer diagnosis. While two previous investigations in a cohort of PFOS-exposed workers suggested a possible positive relation between high PFOS exposure and CRC, the findings were non-significant, based on very small case numbers, and reliant on indirect measures of exposure [14, 15].

Consistent with previous studies, we found the likelihood of CRC diagnosis to increase strongly with age [2]. Menopause was also positively associated with CRC diagnosis, in agreement with findings of some [28‐31] but not other investigations [32‐34]. Likewise, those on lipid-lowering medications were also less likely to have been diagnosed with CRC, similar to findings from some [35‐37], but not all studies [38‐41]. In this study, those with comorbid conditions were more likely to have received a diagnosis of CRC. Previous studies have shown comorbidity to predict earlier stage of CRC diagnosis [42] as well as reduced survival in CRC patients [43]. Several but not all [44] studies have reported increased CRC risk in association with specific conditions, including hypertension, [45, 46] diabetes [45‐47], and other chronic disorders [45, 46], although research specifically assessing the association between overall comorbidity and diagnosed CRC is sparse. BMI was unrelated to CRC diagnosis overall in the current study after adjustment for other sociodemographic and medical factors. However, stratification by gender revealed BMI to be significantly associated with CRC in men but not in women (adjusted ORs for BMI ≥ 25 in men vs. women, respectively = 1.9 (CI 1.1, 3.2) vs. 1.0 (0.6, 1.5), p for interaction = 0.01), findings consistent with those of previous investigations [48].

The strengths of the study include the population-based design, the large sample size, and the high study participation rates in an Appalachian region. Additional strengths include our ability to evaluate persistent biomarkers of PFAA exposure obtained concurrently with survey and medical record information regarding CRC and other chronic conditions, the measurement of a broad array of biomarkers, and the extensive information available on potential confounders and effect modifiers. Ascertainment of CRC was based on participant-reported physician diagnosis and confirmed by chart review. We were also able to assess the association of CRC to PFOA and PFOS over a wide range of serum concentrations, helping to clarify the possible modifying effects of exposure level.

Our study has several important limitations as well. While human half-lives of PFOA and PFOS are long (up to 8.5 [49] and 5.4 years [50], respectively), random misclassification error due to the use of current PFC levels as proxy measures of etiologically relevant exposures may have biased our results toward the null. Most importantly, the cross-sectional nature of the data limits our ability to determine direction of causality. However, the persistence of PFOA and PFOS in human tissues, coupled with the strong inverse associations observed with recent CRC diagnosis in long-term residents, and the robust inverse association of both PFOA and especially, PFOS to CRC at varying exposure levels suggest that a causal relationship may be plausible. While it remains possible that differences in absorption due to the disease or treatment could help explain the observed effects, several lines of reasoning suggest this may not be the case. Adjustment for anemia and for GI symptoms potentially related to reduced absorption did not alter the observed associations, nor did removal of those currently on treatment or who had received chemotherapy. Another substance absorbed primarily through the ileum, folate, showed no relation to CRC diagnosis, and age-adjusted serum folate levels did not differ by disease status. Moreover, the associations of prevalent CRC to serum levels of other PFCs were inconsistent, with 3 of the 5 appearing unrelated or positively related to CRC diagnosis. In addition, CRC has been characterized by reduced gut motility and over-efficient enterohepatic circulation [51], which would be expected to lead to increased, rather than reduced PFAA serum levels [52‐54]. Nonetheless, while these observations collectively suggest that altered PFAA absorption due to CRC and CRC treatment is unlikely to explain the robust inverse associations observed in this study, reverse causality remains a possibility and caution is thus warranted in interpreting our findings.

Additional limitations include lack of information on CRC stage at diagnosis and on certain risk factors for CRC, including inherited genetic alterations, history of inflammatory bowel disease, and specific dietary factors. As only self-reported physician diagnoses of CRC and other cancers were confirmed by chart review, unascertainment due to under-reporting remains possible. However, such underascertainment would be expected to bias the observed associations toward the null, and thus is unlikely to explain our findings. Exclusion of eligible participants with missing data on covariates may have introduced selection bias. Fortunately, the percentage with missing data was small (3.8%). Moreover, given those with missing data did not differ in either their PFC levels or in the incidence of CRC, their exclusion would not be expected to alter the observed associations. Diagnosis could not be validated for a substantial portion (25.8%) of those reporting a physician diagnosis of CRC. However, inclusion of both validated and non-validated cases in the analysis did not alter our findings. Diagnosis was also verified for 100% of participants who reported primary CRC and for whom information was available, indicating excellent reliability and validity of self report diagnosis in this study sample.

Although considerable efforts were made to contact former residents of affected counties, some otherwise eligible adults who moved out of the area may not have participated in the study, possibly introducing sampling bias. Our study population did not include those who had died from CRC and thus may not be representative of adults with more aggressive or advanced disease, limiting generalizability. However, the relation of both PFAAs to CRC diagnosis was stronger in those diagnosed more recently, and remained robust even in those diagnosed within a year of study health assessment, suggesting that survival bias is unlikely to account for our findings. Unmeasured confounding might also help explain our findings, although our ability to control for a large number of both known and potential risk factors for CRC renders this possibility less probable. Finally, some degree of method uncertainty in PFC detection is also possible. However, such uncertainty would be expected to attenuate observed associations and is thus unlikely to explain our findings. In addition, that the laboratory performing the assays followed U.S. Food and Drug Administration (FDA) approved procedures, and assays met the FDA standard for assay precision suggest that significant method uncertainty is unlikely.

If the association with either PFAA proves causal, possible physiologic explanations may include immune modulation, specific anti-cancer effects, including anti-proliferative, apoptotic and differentiating effects, and physiological changes related to the chemical properties of these PFAAs. While animal studies have yielded somewhat inconsistent results [17, 55], a number of investigations in both rodent models and human cell lines suggest PFOA and PFOS can have potent anti-inflammatory effects. For example, in mouse models of inflammation, oral administration of PFOS in doses comparable to those reported in exposed human populations significantly reduced serum TNF-α and decreased peritoneal lavage fluid TNF-α and interleukin-6 levels in response to an immune challenge [17]. Similarly, relatively low doses of PFOA were found to dramatically reduce multiple signs of peripheral inflammation in rats, an effect that did not appear mediated by glucocorticoid receptors [56]. Consistent with these findings, recent in vitro studies of human leukocytes showed PFOS to decrease release of TNF-α following lipopolysaccharide stimulation [57].

The observed anti-inflammatory effects of these PFAAs may be mediated in part by activation of peroxisome proliferator-activated receptors (PPARs). PPARs are transcription factors that belong to the superfamily of nuclear receptors whose natural activating ligands are lipid-derived substrates. A growing body of experimental evidence suggests PFOA and PFOS are potent PPAR ligands, acting as agonists for both the alpha (PPAR-α) and gamma isotypes (PPAR-γ) [17, 18, 58‐60]. PPARs are expressed on macrophages [61], and are highly expressed in intestinal [59] and other tissues [18]. These receptors are now thought to play a major role in the regulation of inflammatory responses [11, 18, 62‐64]. For example, PPAR-γ has been shown to down-regulate the expression of inflammatory cytokines, inhibit major inflammatory signaling pathways [65], and direct immune cell differentiation towards anti-inflammatory phenotypes [66].

PPARs are also thought to play a key role in the differentiation, apoptosis and proliferation of colon and other tumor cells [10, 11, 63], contributing to the growing research interest in these receptors and their ligands as potential chemoprevention and chemotherapeutic agents [10, 11]. Studies in rodent models of colorectal and other tumors, as well as in human normal and tumor cell lines have shown PPAR-γ ligands to exert powerful anti-cancer effects; these effects appear mediated by the differentiating, pro-apoptotic, and anti-proliferative effects on malignant cells, as well as by the anti-inflammatory actions of PPAR-γ [10, 11]. Likewise, PPAR-α has also been implicated in the inception and progression of CRC. For example, PPAR-α ligands induce apoptosis in human tumor cell lines and PPAR-α expression is reduced in human tubular adenomas and colonic adenocarinoma cells compared to normal human colonic mucosa [63, 67]. In addition, both PPAR-α and PPAR-γ ligands have been shown to suppress polyp formation in an animal model of familial polyposis [67, 68].

Finally, PFOS and PFOA are also surfactants, characterized by strong hydrophobic properties [69], which could, in turn, contribute to a protective effect against CRC. While this possibility remains unexplored in PFAAs, other compounds with hydrophobic properties have been shown to induce cell cycle arrest and apoptosis of human colon cancer [70] and other tumor cells [71].

In this cross-sectional study of a large Appalachian population, we found a strong inverse linear association between PFOS and likelihood of CRC diagnosis and a significant, although more modest inverse association between PFOA and diagnosis of CRC. If confirmed in prospective investigations, these findings could aid in identifying new strategies for CRC prevention and treatment and help inform future studies regarding possible mechanisms underlying the pathogenesis of this of this common and often fatal cancer.